PROJECT SUMMARY Bile acid (BA) signaling pathways are plausible new therapeutic targets for metabolic disease. One potential approach is to improve the composition of endogenous BAs. Humans have dozens of species of BAs, and each is differently able to carry out BA functions. We have previously identified a subset of BAs?those containing a hydroxyl group at the carbon 12 position?that are regulated by hepatic insulin signaling, increased in human insulin resistance, and correlated with metabolic dysfunctions. Eliminating 12-hydroxy BAs by ablating the enzyme required for their synthesis, Cyp8b1, lowers body weight, lipids, and glucose. However the molecular mechanisms of these improvements are incompletely understood, particularly the mechanisms leading to improved glycemia. Thus we have identified three fundamental, unanswered questions for our studies: First, what signaling pathways are required to bring about these improvements? Second, what are the direct effects of 12-hydroxy BAs on cellular metabolic and signaling cascades? Third, what are the metabolic benefits of eliminating 12-hydroxy BAs in pre-existing diabetes/obesity? These questions will be addressed in vivo and ex vivo, using genetic, biochemical, and humanized tools. The proposed studies will improve our understanding of this conserved enzyme in physiology and its potential as a new therapeutic strategy.